1702 CHEMICAL PATH OF CARBON DIOXIDE REDUCTION CHAP. 36 



This may be the place to mention the study, by means of the C(14) tracer, of the 

 de-acidification of succulents in Ught— a phenomenon described in chapter 10 (section 

 D2). The two alternative interpretations suggested there, were: (1) "photosynthesis" 

 with malic (or citric) acid as "substitute oxidant" (replacing CO2 as hydrogen acceptor), 

 and (2) photoxidation of these acids to carbon dioxide, followed by normal photosynthe- 

 sis. Tracer experiments could perhaps permit a choice between these two hypotheses. 



Thurlow and Bonner (1948) and Varner and Burrell (1950) found that, in darkness, 

 acidification in tagged C*02 produced, in Bryophyllum calycinum, malic acid tagged 

 preferentially in the carboxyl group, indicating its probable formation by the Wood- 

 Werkman reaction (i. e., carboxylation of pyruvic to oxalacetic acid followed by reduction 

 of the latter to mahc acid. It will be recalled that the same mechanism has been discussed 

 — but more recently abandoned — by Calvin and co-workers also as explanation of the 

 tagging of malic acid in photosynthesis.) 



In fight, the hexoses which are formed in the succulent when malic acid disappears, 

 were tagged preferentially in the 3,4 position (as in ordinary photosynthesis, cf. table 

 36. VII). This indicates that the C* dicarboxyfic acid is either photoxidized completely 

 to CO2, or, at least, decarboxylated to a C3 acid (with the remaining carboxyl still tagged) 

 before it is used for the synthesis of sugars by the reduction of the C3 acid to triose and 

 head-on condensation of two triose molecules to a hexose (leading to the accumulation of 

 labelled carbon in the two middle atoms of the Ce chain). 



If mafic acid were to take direct part in Ce synthesis, e. g., in the way postulated by 

 Benson, Calvin et al. in 1949-1950 {i. e., spfitting of the C4 chain into two C2 fragments, 

 and their carboxylation), this would lead to a C3 acid with labelled carbon in the /3- 

 position, rather than in the carbo.xyl; reduction of the product to triose and condensation 

 of the latter to hexose would then place the labels on carbon atoms 2 and 3 in the Ce 

 chain. 



Stutz (1950) exposed Bryophyllum calycinum to labelled carbon dioxide for several 

 hours in light and chromatographed the synthesized organic acids on a column ; activity 

 was found in succinic, oxafic, malic, citric, and iso-citric acid. After 12 hours in light 

 and 12 hours in darkness, almost 90% of C* was found in mafic acid, with only 58% in 

 iso-citric acid and 4% in citric acid (although the absolute amount of iso-citric acid 

 present was larger than that of malic acid). Similar preferential labelling of mafic acid 

 was noted after V2, 2 or 4 hours in fight without a subsequent dark period. When Cali- 

 cynum leaves, exposed to C*02for 15 min. in light, were left in darkness (without C*02) 

 for 4 hours (to observe the shift in the relative concentration of the different acids, cf. 

 p. 269), the specific activity of all acids was found to increase (obviously at the cost of 

 some nonacid C* reservoir formed in light), despite a strong increase in the absolute 

 quantity of all acids. (Mafic acid increased, in the dark period, by a factor of 5 in abso- 

 lute amount and a factor of 2 in specific activity, citric acid by a factor of 3 in quantity 

 and 6 in specific activity, iso-citric acid by a factor of 2.8 in quantity and 2.4 in specific 

 activity.) Similar experiments were carried out with tobacco leaves (which also store 

 malic and citric acid, cf. p. 264). There, too, C* was fixed predominantly in malic acid. 

 It was shown that 100 mg. quantities of heavily labelled mafic and citric acid can be pre- 

 pared by growing Bryophyllum or tobacco seedlings in C*( 14)02. 



B. Photosynthesis and Phosphate Metabolism* 



As described in part A, most reactions in the reduction of carbon dioxide 

 in photosynthesis involve phosphate esters rather than free organic com- 



* Bibliography, page 1712. 



